The long-term objective of this project is to understand the mechanisms by which neurons control the voltage-activated Ca2+ currents in their surface membranes. These Ca2+ currents are directly responsible for the intracellular Ca2+ signals that trigger neurotransmitter release, modulate membrane excitability and synaptic efficacy, and control neurite growth. The project is focused on two phenomena that strongly change the function of Ca2+ channels, the "block" of Ca2+ channels by intracellular Ca2+ and the "rundown" of Ca2+ channels when exposed to artificial internal environments. The goal is to determine the molecular mechanisms underlying these phenomena, testing the hypothesis that common mechanisms underlie both. During the first period of this project it was found that submicromolar concentrations of Ca2+ rapidly blocked Ca2+ channels, indicating few molecular steps between Ca2+ binding and channel block. Both block by intracellular Ca2+ and rundown were found to involve the local cytoskeleton and not to depend on dephosphorylation, as has been commonly assumed. The proposal for the next period of this project is to further characterize the interaction between cytoskeleton and Ca2+ channels and to more reliably quantify the relation between intracellular Ca2+ and channel function. The role of the cytoskeleton in Controlling Ca2+ channels under more physiological conditions (without rundown) will be studied, and cytoskeletal proteins involved in Ca2+ channel control will be identified. Photolyzable Ca2+ chelators and ratiometric Ca2+ monitoring will be used to complete the quantitative study of the relationship between intracellular Ca2+ and channel function. Patch-clamp physiological techniques will be applied to acutely isolated neurons from the snail Lymnaea. Rundown will primarily be studied in inside-out patches, so that non-permeant drugs, enzymes and other proteins can be applied with certainty. Studies of Ca2+ channel block by intracellular Ca2+ will be done in two ways: direct perfusion of inside- out patches with solutions of well-buffered Ca2+ concentrations, or flash photolysis of Ca2+ chelators (DM-nitrophen, nitrophenyl-EGTA, diazo-4) in whole cells, while monitoring intracellular Ca2+ with fluorescent probes (fura-2, furaptra). This project will provide direct measurements of two of the most important components of neuronal Ca2+ regulation, the cell's ability to handle Ca2+ released inside the cell and the effect of intracellular Ca2+ on membrane Ca2+ influx. These components of Ca2+ regulation are central to understanding the Ca2+-induced cell death common to human pathologies as diverse as cerebral stroke and Alzheimer's disease.